Linker and support for solid phase synthesis of nucleic acid

ABSTRACT

The invention provides a universal linker capable of synthesizing nucleic acid having a phosphate group at the 3′ terminal, a universal support carrying the linker, and a synthesis method of nucleic acid using the universal support. The linker for solid phase synthesis of nucleic acid contains a compound represented by at least one of the following formulae 
     
       
         
         
             
             
         
       
     
     wherein each symbol is as defined in the specification.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a linker to be used for solid phase synthesis of nucleic acid, a support for solid phase synthesis carrying the linker, and a production method of nucleic acid using the support.

BACKGROUND OF THE INVENTION

For chemical synthesis of nucleic acid such as DNA, RNA and the like, a solid phase synthesis process using a phosphoramidite method is widely employed. In the solid phase phosphoramidite method, nucleic acid is generally synthesized by the following steps.

First, nucleoside to be the 3′ terminal of the nucleic acid to be synthesized is ester bonded to a cleaving linker such as succinyl group and the like via 3′-OH group so that the nucleoside is previously carried on a support for solid phase synthesis (nucleoside linker). Then, the support for solid phase synthesis on which the nucleoside linker is carried is placed in a reaction column which is then set on an automated nucleic acid synthesizer.

Thereafter, a synthesis reaction comprising the following steps is generally performed in the reaction column according to a synthesis program of the automated nucleic acid synthesizer:

-   (1) a step of deprotection of 5′-OH group of the protected     nucleoside with an acid such as trichloroacetic acid/dichloromethane     solution and the like; -   (2) a step of coupling nucleosidephosphoramidite (nucleic acid     monomer) with the deprotected 5′-OH group in the presence of an     activator (tetrazole etc.); -   (3) a step of capping an unreacted 5′-OH group with acetic anhydride     and the like; and -   (4) a step of oxidizing phosphite with aqueous iodine and the like.     By repeating the synthesis cycle, an elongation reaction of     oligonucleotide from the 3′ terminal to the 5′ terminal direction is     promoted, and a nucleic acid having a desired sequence is     synthesized.

Lastly, a cleaving linker is hydrolyzed with aqueous ammonia, methylamine solution and the like to cleave the synthesized nucleic acid from the support for solid phase synthesis (non-patent document 1).

When the above-mentioned synthesis is performed, as mentioned above, it is necessary to carry, in advance, nucleoside to be the 3′ terminal (starting material) on a support for solid phase synthesis via a cleaving linker. Moreover, the 3′ terminal varies depending on the sequence of nucleic acid desired to be synthesized. In the case of DNA oligonucleotide, 4 kinds of dA, dG, dC, dT are necessary, and in the case of RNA, 4 kinds of rA, rG, rC, rU are also necessary. For synthesis of modified oligonucleotide, a support for solid phase synthesis previously carrying a modified nucleoside is necessary, making the process complicated.

To solve the aforementioned problems, a support for solid phase synthesis carrying a universal linker (universal support) has been developed as a linker to connect a solid phase support and a starting material, in the place of nucleoside•succinyl linker and the like generally used heretofore. Using the universal support, the process includes, irrespective of the kind of nucleoside or nucleotide for the 3′ terminal of nucleic acid desired to be synthesized, reacting nucleoside phosphoramidite to be the 3′ terminal in the same step as general automated nucleic acid synthesis to start the synthesis and, after synthesizing the desired nucleic acid, cleaving the nucleic acid from the support for solid phase synthesis by a method similar to a general method. It is not necessary to prepare a support for solid phase synthesis carrying various nucleoside-linkers as mentioned above.

There are proposed some universal supports, which render the 3′ terminal of the nucleic acid desired to be synthesized an —OH group (patent documents 1-4 and non-patent documents 2 and 3). The structure of these universal supports has two adjacent carbon atoms, one carbon atom being bound with —OH group to be the starting point of nucleic acid synthesis, and the other carbon atom being bound with a group (e.g., —OH group, —NH₂ group, —SH group) to be a nucleophilic group upon removal of the protecting group. When the nucleic acid is cleaved by aqueous ammonia and the like after the nucleic acid synthesis, the protecting group of these nucleophilic groups are also dissociated to attack the 3′ terminal phosphorus, and the phosphate group is cleaved from the 3′ terminal to form cyclophosphoric acid. All are used to synthesize nucleic acid having an —OH group at the 3′ terminal.

In addition, some universal supports have been proposed for providing a phosphate group at the 5′ terminal or 3′ terminal of nucleic acid desired to be synthesized. For example, patent document 5 and non-patent document 4 disclose a universal linker capable of synthesizing nucleic acid having a phosphate group at the 5′terminal, a universal support carrying the universal linker, and a synthesis method of nucleic acid using the universal support.

Since a nucleic acid having a phosphate group at the 5′ terminal or 3′ terminal can be widely used in various fields of biochemistry such as chemical linkage reaction of nucleic acid, modification of nucleic acid utilizing terminal phosphate group, structural study of nucleic acid and the like, it is extremely useful. In view of such situation, a universal linker capable of synthesizing a nucleic acid having a phosphate group at the 5′ terminal or 3′ terminal, and a universal support carrying the linker are desired.

PRIOR ART [Patent Documents]

-   patent document 1: U.S. Pat. No. 5,681,945 -   patent document 2: U.S. Pat. No. 6,653,468 -   patent document 3: WO2005/049621 -   patent document 4: US-A-2005/0182241 -   patent document 5: U.S. Pat. No. 5,959,090

[Non-Patent Documents]

-   non-patent document 1: Current Protocols in Nucleic Acid Chemistry     (2000) -   non-patent document 2: Bio Techniques, 22, 752-756 (1997) -   non-patent document 3: Tetrahedron, 57, 4977-4986 (2001) -   non-patent document 4: Tetrahedron Letters, 27: 4705-4708 (1986)

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

The present invention aims to provide a universal linker capable of synthesizing a nucleic acid having a phosphate group at the 3′ terminal, a universal support carrying the linker, and a method for synthesizing a nucleic acid using the universal support.

Particularly, the present invention aims to provide a support for solid phase synthesis, which is capable of universally synthesizing a nucleic acid having a phosphate group at the 3′ terminal in a high purity, which does not require a support for solid phase synthesis with modified nucleoside carried in advance even when synthesizing a modified oligonucleotide.

Means of Solving the Problems

Accordingly, the present invention provides the following.

-   [1] A linker for solid phase synthesis of nucleic acid, comprising a     compound represented by at least one of the following formulae

wherein

X is a hydroxyl-protecting group dissociated by acid,

L is a linkage moiety cleaved by alkali, and

R¹, R², R³ and R⁴ are each independently a hydrogen atom, an alkyl group having a carbon number of 1-6, an alkoxy group having a carbon number of 1-6, an alkylamino group having a carbon number of 1-6 or a halogen atom.

-   [2] The linker for solid phase synthesis of nucleic acid according     to the above-mentioned [1], wherein L is a succinyl group. -   [3] The linker for solid phase synthesis of nucleic acid according     to the above-mentioned [1] or [2], wherein X is a dimethoxytrityl     group. -   [4] A support for solid phase synthesis of nucleic acid, comprising     a structure shown by at least one of the following formulae

wherein

X is a hydroxyl-protecting group dissociated by acid,

L is a linkage moiety cleaved by alkali,

Sp is a solid phase support, and

R¹, R², R³ and R⁴ are each independently a hydrogen atom, an alkyl group having a carbon number of 1-6, an alkoxy m group having a carbon number of 1-6, an alkylamino group having a carbon number of 1-6 or a halogen atom.

-   [5] The support for solid phase synthesis of nucleic acid according     to the above-mentioned [4], wherein L is a succinyl group. -   [6] The support for solid phase synthesis of nucleic acid according     to the above-mentioned [4] or [5], wherein X is a dimethoxytrityl     group. -   [7] The support for solid phase synthesis of nucleic acid according     to any of the above-mentioned [4] to [6], wherein Sp is a solid     phase support comprised of porous synthetic polymer particles or     porous glass particles. -   [8] A method of producing a nucleic acid, comprising a step of     performing a nucleic acid synthesis reaction on the support for     solid phase synthesis of nucleic acid according to any of the     above-mentioned [4] to [7]. -   [9] The production method of the above-mentioned [8], wherein the     nucleic acid synthesis reaction is performed by a solid phase     phosphoramidite method.

Effect of the Invention

The linker for solid phase synthesis of nucleic acid of the present invention is a universal linker, and the support for nucleic acid synthesis of the present invention, carrying the linker is a universal support. The support for nucleic acid synthesis carrying the linker of the present invention does not require a support for solid phase synthesis carrying a nucleotide to be the 3′ terminal of the nucleic acid desired to be synthesized, and can synthesize a nucleic acid having a phosphate group introduced into the 3′ terminal at a high purity. It does not require a support for solid phase synthesis with a modified nucleoside carried in advance even when synthesizing a modified oligonucleotide, and can synthesize a nucleic acid having a phosphate group introduced into the 3′ terminal at a high purity.

Therefore, the support for nucleic acid synthesis of the present invention is preferably used for automatic synthesis of a nucleic acid having a phosphate group at the 3′ terminal. Furthermore, the production method of a nucleic acid using the support for nucleic acid synthesis of the present invention does not require separate introduction of a phosphate group into the 3′ terminal of a synthesized nucleic acid, unlike conventional methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows HPLC chart of the DNA oligonucleotide solutions obtained in Example 2 (A), Comparative Example 1 (B), Example 3 (C) and Comparative Example 2 (D).

FIG. 2 shows HPLC chart of the DNA oligonucleotide solutions obtained in Example 5 (A), Comparative Example 3 (B), Example 6 (C) and Comparative Example 4 (D).

MODE FOR CARRYING OUT THE INVENTION

The term “nucleic acid” as used herein refers to a linear compound (oligonucleotide) wherein nucleotides are connected via phosphodiester bonds, and is understood to encompass DNA, RNA and the like. The nucleic acid may be single-stranded or double-stranded. Since it allows an efficient synthesis using a nucleic acid synthesizer, the nucleic acid is preferably single-stranded. The “nucleic acid” in the present specification includes not only a oligonucleotide containing a purine base such as adenine (A), guanine (G) and the like and a pyrimidine base such as thymine (T), cytosine(C), uracil (U) and the like but also a modified oligonucleotide containing other modified heterocyclic base.

The nucleotide length of the nucleic acid is not particularly limited, and the nucleic acid is preferably 2 to 200 nucleotides long. If the nucleic acid is too long, the yield and purity of the nucleic acid obtained decrease.

The “linker” in the present specification refers to a molecule that links two substances via a covalent bond. In the present invention, the linker connects a solid phase support and a nucleic acid.

The linker for solid phase synthesis of nucleic acid of the present invention is represented by the following formula.

wherein:

-   X is a hydroxyl-protecting group dissociated by acid. Examples of     the protecting group include a trityl group (Tr), a     monomethoxytrityl group (MMTr), a dimethoxytrityl group (DMTr) and     the like. These protecting groups can be dissociated using a     dichloromethane or toluene solution of Bronsted acid such as     trichloroacetic acid, dichloroacetic acid and the like. Since     deprotection with an acid is easy, DMTr is most preferably used.

L is a linkage moiety cleaved by alkali, and examples thereof include, but are not limited to, a succinyl group (succinyl linker), Q linker (Pon et al., Nucleic Acids Res., 27, 1531 (1999)) and the like. A succinyl linker is preferably used. These linkage moieties can be easily cleaved by hydrolysis with aqueous ammonia, aqueous ammonia/methylamine mixture and the like, and oligonucleotide is cleaved from a support for solid phase synthesis after completion of automatic synthesis. That is, when a molecule with synthesized oligonucleotide bound at the X position is produced, it is considered that L is cleaved from the linker for solid phase synthesis of nucleic acid of the present invention, which is shown by the above-mentioned formula, simultaneously with which the linker of the present invention is released from the synthetic oligonucleotide due to the nucleophilic reaction of “O⁻” produced at the ortho-position or para-position, whereby a synthetic oligonucleotide having a phosphate group at the 3′ terminal can be obtained. The reaction as such universal support of the present invention is described later.

R¹, R², R³ and R⁴ are each independently a hydrogen atom, an alkyl group having a carbon number of 1-6, an alkoxy group having a carbon number of 1-6, an alkylamino group having a carbon number of 1-6 or a halogen atom. Examples of the alkyl group having a carbon number of 1-6 include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an n-amyl group, an isoamyl group, a sec-amyl group, a tert-amyl group, a hexyl group and the like. Among these, a methyl group or an ethyl group is preferable. Examples of the alkoxy group having a carbon number of 1-6 include a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a pentyloxy group, a hexyloxy group and the like. Among these, a methoxy group is preferable. Examples of the alkylamino group having a carbon number of 1-6 include a methylamino group, a dimethylamino group, an ethylamino group, a diethylamino group, a propylamino group, a dipropylamino group, an isopropylamino group, a diisopropylamino group and the like. Among these, a dimethylamino group is preferable. In addition, examples of the halogen atom include chlorine, fluorine, bromine, iodine and the like. Among these, chlorine or fluorine is preferable.

The solid phase support for solid phase synthesis of nucleic acid of the present invention carries the above-mentioned linker for solid phase synthesis of nucleic acid of the present invention, and is represented by the following formula

wherein X and L, and R¹, R², R³ and R⁴ are as defined above.

Sp is a solid phase support. The solid phase support is not particularly limited as long as a reagent used in excess can be easily removed by washing and, for example, glass porous support, porous synthetic polymer support such as polystyrene support, acrylamide support etc., and the like can be mentioned.

In a preferable embodiment of the present invention, in the above-mentioned formula, Sp is a glass porous support. Here, the “glass porous support” refers to a porous support containing glass as a constituent component and examples thereof include, but are not limited to, porous glass particles in a granular shape (CPG) and the like. More specifically, as the aforementioned CPG, a CPG solid phase support having a long chain aminoalkyl spacer (LCAA-CPG solid phase support) is preferably used, and further, for the synthesis of a long chain nucleotide, one having a CPG pore of preferably 20-400 nm, more preferably 50-200 nm, most preferably 100 nm, is used.

In another preferable embodiment of the present invention, in the above-mentioned formula, Sp is a polystyrene support. Here, the “polystyrene support” is a solid phase support constituted with a copolymer containing structural unit (A) represented by the following formula:

and/or a substituted derivative thereof as a structural unit. Examples of the substituted derivatives of the structural unit (A) include a compound wherein one or more hydrogen atoms contained in the structural unit (A) (including hydrogen atom of benzene ring) are substituted by an alkyl group having a carbon number of 1-6 (e.g., methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-amyl group, isoamyl group, sec-amyl group, tert-amyl group), a halogen atom, an amino group, a hydroxy group, a carboxyl group, a sulfone group, a cyano group, a methoxy group, a nitro group, a vinyl group and the like. The substituent is preferably an amino group or a hydroxy group. The position of the substituent is not particularly limited, and is preferably the para-position with respect to the main chain on the benzene ring. A preferable substitution derivative of the structural unit (A) is the hydroxystyrene structural unit (B) shown in the following formula.

The amount of structural unit (A) and/or substitution derivatives thereof relative to the total amount of the structural units contained in the copolymer constituting the polystyrene support is not particularly limited, and is generally 50 to 100% by weight, preferably 60 to 100% by weight.

Specific examples of the monomer compound copolymerizable with the above-mentioned structural unit (A) include, but are not limited to, styrene; nucleus alkyl substituted styrene such as o-methylstyrene, m-methylstyrene, p-methylstyrene, 2,4-dimethylstyrene, ethylstyrene, trimethylstyrene, and p-t-butylstyrene and the like; α-alkyl substituted styrene such as α-methylstyrene, α-methyl-p-methylstyrene and the like; nucleus halogenated styrene such as chlorostyrene, dichlorostyrene, fluorostyrene, pentafluorostyrene, bromostyrene and the like; alkyl halide styrene such as chloromethylstyrene, fluoromethylstyrene and the like; hydroxystyrene; hydroxymethylstyrene; vinyl benzoate; sodium styrenesulfonate; cyanostyrene; methoxystyrene; ethoxystyrene; butoxy styrene; nitrostyrene; acyloxy styrene such as acetoxystyrene, benzoxystyrene etc., and the like. Further examples include, but are not limited to, alkyl (meth)acrylate monomer such as methyl(meth)acrylate, ethyl(meth)acrylate, propyl(meth)acrylate, butyl(meth)acrylate and the like, vinyl cyanide monomer such as acrylonitrile, methacrylonitrile, ethacrylonitrile etc., and the like.

In another embodiment of the present invention, in the above-mentioned formula, Sp may be a solid phase support consisting of styrene-hydroxystyrene-divinylbenzene copolymer particles (JP-A-2005-097545, JP-A-2005-325272 and JP-A-2006-342245) or a styrene-(meth)acrylonitrile-hydroxystyrene-divinylbenzene copolymer (JP-A-2008-074979), having, in addition to the above-mentioned structural units (A) and (B), divinylbenzene structural unit (C) represented by the following formula:

and the like. In addition, one or more hydrogen atoms (including hydrogen atom of benzene ring) in the above-mentioned divinylbenzene structural unit (C) may be substituted by an alkyl group having a carbon number of 1-6 (e.g., methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-amyl group, isoamyl group, sec-amyl group, tert-amyl group), a halogen atom, an amino group, a hydroxy group, a carboxyl group, a sulfone group, a cyano group, a methoxy group, a nitro group, a vinyl group and the like. The substituent is preferably an amino group or a hydroxy group.

In the above-mentioned formula, when Sp is a solid phase support comprised of the aforementioned styrene-(meth)acrylonitrile-hydroxystyrene-divinylbenzene copolymer, the structural unit of (meth)acrylonitrile may contain each or both of the structural units of acrylonitrile and methacrylonitrile. When the amount of the structural unit of (meth)acrylonitrile to the total amount of the structural unit of the styrene-(meth)acrylonitrile-hydroxystyrene-divinylbenzene copolymer is too high or too low, the degree of swelling greatly varies depending on the kind of organic solvent. Therefore, the amount is preferably 2-11 mmol/g.

In another preferable embodiment of the present invention, in the above-mentioned formula, Sp is an acrylamide support. More specifically, in the above-mentioned formula, Sp may be a support for solid phase synthesis, which is comprised of, in addition to the above-mentioned structural units (A) and (C), an aromatic monovinyl compound-divinyl compound-(meth)acrylamide derivative copolymer further containing a (meth)acrylamide derivative monomer.

In the above-mentioned formula, when Sp is a solid phase support comprised of the aforementioned aromatic monovinyl compound-divinyl compound-(meth)acrylamide derivative copolymer, the aforementioned aromatic monovinyl compound is not particularly limited. For example, styrene; nucleus alkyl substituted styrene such as o-methylstyrene, m-methylstyrene, p-methylstyrene, 2,4-dimethylstyrene, ethylstyrene, trimethylstyrene, p-t-butylstyrene and the like; α-alkyl substituted styrene such as α-methylstyrene, α-methyl-p-methylstyrene and the like; nucleus halogenated styrene such as chiorostyrene, dichlorostyrene, fluorostyrene, pentafluorostyrene, bromostyrene and the like; alkyl halide styrene such as chloromethylstyrene, fluoromethylstyrene and the like; hydroxystyrene; hydroxymethylstyrene; vinyl benzoate; sodium styrenesulfonate; cyanostyrene; methoxystyrene; ethoxystyrene; butoxy styrene; nitrostyrene; acyloxystyrene such as acetoxystyrene, benzoxystyrene etc., and the like can be mentioned, with preference given to styrene.

Examples of the aforementioned (meth)acrylamide derivative include, but are not limited to, N-alkyl(meth)acrylamide; N,N-dialkyl(meth)acrylamide; N-alkoxyalkyl(meth)acrylamide; 2-(meth)acrylamide alkane sulfonic acid; N-alkylol(meth)acrylamide such as N-methylol(meth)acrylamide and the like; acrylamide; methacrylamide; diacetone acrylamide; N,N-dimethyl aminopropylacrylamide; acryloylmorpholine; N-phenoxymethyl(meth)acrylamide and the like.

In addition, the alkyl contained in the aforementioned N-alkyl(meth)acrylamide is generally a linear or branched alkyl having a carbon number of 1-6, preferably 1-3. Examples of the N-alkyl(meth)acrylamide include N-methyl(meth)acrylamide, N-ethyl(meth)acrylamide, N-n-propyl(meth)acrylamide, N-isopropyl(meth)acrylamide, N-tert-butyl(meth)acrylamide, and N-lauryl(meth)acrylamide and the like.

The two alkyls contained in the aforementioned N,N-dialkyl(meth)acrylamide are each generally a linear or branched alkyl having a carbon number 1-6, preferably 1-3. Examples of the N,N-dialkyl(meth)acrylamide include N,N-dimethyl(meth)acrylamide, N,N-diethyl(meth)acrylamide, N,N-diisopropyl(meth)acrylamide, N,N-di-tert-butyl(meth)acrylamide, N,N-dilauryl(meth)acrylamide, N,N-di-tert-octyl(meth)acrylamide, N,N-dilauryl(meth)acrylamide, N,N-dicyclohexyl(meth)acrylamide and the like.

The alkoxy contained in the aforementioned N-alkoxyalkyl(meth)acrylamide is generally a linear or branched alkoxy having a carbon number of 1-6, preferably 1-3. The alkyl contained in N-alkoxyalkyl(meth)acrylamide is generally a linear or branched alkyl having a carbon number of 1-6, preferably 1-3. Examples of the N-alkoxyalkyl(meth)acrylamide include N-methoxymethyl(meth)acrylamide, N-ethoxymethyl(meth)acrylamide, N-propoxymethyl(meth)acrylamide, N-butoxy methyl(meth)acrylamide, N-ethoxyethyl(meth)acrylamide, N-methoxypropyl(meth)acrylamide, N-ethoxypropyl(meth)acrylamide, N-isopropoxyethyl(meth)acrylamide and the like.

The alkane contained in the aforementioned 2-(meth)acrylamide alkanesulfonic acid is generally a linear or branched alkane having a carbon number of 1-6, preferably 1-3. Examples of the 2-(meth)acrylamide alkanesulfonic acid include 2-acrylamide propanesulfonic acid, 2-acrylamide-n-butanesulfonic acid, 2-acrylamide-n-hexanesulfonic acid, 2-acrylamide-2-methylpropanesulfonic acid and the like.

The aforementioned (meth)acrylamide derivative is preferably diacetone acrylamide, N-isopropyl(meth)acrylamide, N-methoxymethyl(meth)acrylamide or N,N,-dimethyl(meth)acrylamide.

A typical structural unit derived from (meth)acrylamide derivative monomer includes the following structural units.

In the above-mentioned formula, when Sp is an acrylamide solid phase support and the content of the structural unit derived from a (meth)acrylamide derivative monomer is too small, an effect of avoiding a decrease in the synthesis amount of nucleic acid and a decrease in the synthesis purity cannot be obtained. On the other hand, when it is too high, a porous resin bead is difficult to form. Therefore, it is preferably 0.3-4 mmol/g, more preferably 0.4-3.5 mmol/g, further preferably 0.6-3 mmol/g.

The porous particle to be used as the support for solid phase synthesis of the present invention is preferably one having a functional group that contributes to nucleic acid synthesis. The “contributes to nucleic acid synthesis” refers to a functional group which can be the starting point of nucleic acid synthesis and to which a linker can be added. Specifically, an amino group, a hydroxy group and the like can be mentioned.

The content of the functional group that contributes to nucleic acid synthesis is not particularly limited. When the content of the functional group is too small, the yield of nucleic acid decreases and when it is too high, the purity of the obtained nucleic acid decreases. Therefore, it is preferably 10-2000 μmol/g, more preferably 50-1000 μmol/g, further preferably 100-800 μmol/g.

When the functional group that contributes to nucleic acid synthesis is a hydroxyl group, the amount of the hydroxyl group of the porous particles of the present invention is measured by titration based on JIS K0070. Specifically, an acetylation reagent is produced by adding pyridine to 25 g of acetic anhydride to the total amount of 100 mL. The above-mentioned acetylation reagent (0.5 mL), pyridine (4.5 mL) and a sample (about 0.5 g) are placed in a flask, and the mixture is heated at 95-100° C. for 2 hr to acetylate the hydroxyl group. Then, distilled water (1 mL) is added into the flask, and the mixture is heated to decompose acetic anhydride that was not consumed by acetylation to acetic acid. The amount of the acetic acid is measured by neutralization titration using 0.5 mol/L of aqueous potassium hydroxide solution. Separately, a blank measurement is performed in the same way, but without adding the sample. Since the difference in the number of moles in the above-mentioned two measurements is the number of moles of acetic anhydride consumed by acetylation of the hydroxyl group of the sample (i.e., amount of hydroxyl group of the sample), this value is divided by the sample weight to determine the amount of the hydroxyl group per 1 g of the sample.

The amount of the linker to be bound to the solid phase support is not particularly limited. When the linker content is too low, the yield of nucleic acid decreases. When the linker content is too high, the purity of the nucleic acid obtained tends to decrease and the number of nucleotides of the obtained nucleic acid tends to decrease. Therefore, it is preferably within the range of 20-800 μmol/g, more preferably 25-500 μmol/g.

The shape of the aforementioned porous solid phase support is not particularly limited and may be any shape of plate, particle, fiber and the like. Since the packing efficiency to a synthesis reaction container can be enhanced, and the reaction container is not easily broken, a porous synthetic polymer having a particle shape is preferable. The term “particle” in the specification does not mean being exactly spherical, but means having any constant form (e.g., roughly spherical forms such as ellipse spherical, polygonal form, cylindrical form, irregular forms such as konpeito form, and the like). While the size (volume) of one particle of the porous synthetic polymer particles is not particularly limited, when the average particle size measured by laser diffraction (scattering type) of the porous particles is smaller than 1 μm, inconvenience occurs when it is packed in a column and used in that the back pressure becomes too high or a solution sending rate becomes slow. On the other hand, when the average particle size is more than 1000 μm, the gap between the support particles becomes large and efficient packing of support particles in a column having a predetermined volume becomes difficult. Therefore, it is preferably 1-1000 μm, more preferably 5-500 μm, further preferably 10-200 μm.

While the specific surface area of the aforementioned synthetic polymer particles measured by a multi-point BET method is not particularly limited, when the specific surface area is less than 0.1 m²/g, the degree of swelling in an organic solvent becomes low, and a synthesis reaction tends to be difficult to occur. On the other hand, when it is more than 500 m²/g, fine pore size becomes small, and a synthesis reaction tends to be difficult to occur. Therefore, the specific surface area is preferably 0.1-500 m²/g, more preferably 10-300 m²/g, further preferably 50-200 m²/g.

In addition, the average fine pore size of the aforementioned synthetic polymer particles measured by a mercury intrusion technique is not particularly limited. However, when the pore size is too small, the field of the synthesis reaction becomes small and a desired reaction does not occur easily, or the nucleotide length tends to be less than a desired number. On the other hand, when the pore size is too large, the frequency of contact between a hydroxyl group and a substance involved in the reaction on the surface of polymer particles, which is the reaction field, decreases to lower the yield. Therefore the average fine pore size is preferably 1-200 nm, more preferably 5-100 nm, more preferably 20-70 nm.

In a preferable embodiment of the solid phase support of the present invention, in the above-mentioned formula, Sp is a low swelling cross-linking polystyrene particle commercially available as NittoPhase (registered trade mark) (manufactured by NITTO DENKO Co., Ltd.). A solid phase nucleic acid synthesis method using NittoPhase (registered trade mark) is preferably used since it shows a small peak area due to impurity and guarantees high yield and high purity in a wide scale from labo scale to mass synthesis system.

Production Method of Support for Solid Phase Synthesis of Nucleic Acid of the Present Invention

While the production method of the support for solid phase synthesis of nucleic acid of the present invention is not particularly limited, for example, the support can be produced by the following method.

An aromatic ring compound having a hydroxy group and a hydroxymethyl group at the ortho-position or para-position (e.g., 2-hydroxybenzyl alcohol, 4-hydroxybenzyl alcohol) is reacted with 4,4′-dimethoxytrityl chloride and the like to protect the hydroxymethyl group. Then, this compound is reacted with succinic anhydride together with triethylamine to bind a succinyl linker moiety to a hydroxy group, which is then bound with a solid phase support having —OH group or —NH₂ group to give the support for solid phase synthesis of nucleic acid of the present invention.

Synthesis Method of Nucleic Acid Using Support for Solid Phase Synthesis of Nucleic Acid of the Present Invention

For nucleic acid synthesis using the support for solid phase synthesis of nucleic acid of the present invention, a nucleic acid automatic synthesizer is used and various synthesis methods known per se can be used. In the present specification, the “nucleic acid synthesis reaction” particularly means an elongation reaction of nucleotide constituting a nucleic acid. Hence, nucleotides are sequentially bound to a nucleoside, nucleotide or oligonucleotide bound to the solid phase support, whereby an elongated oligonucleotide is obtained. The nucleic acid synthesis reaction can be performed by the H-phosphonate method, phosphoester method, solid phase phosphoramidite method and the like. Of these, since high capacity of synthesizing nucleic acid and high purity of nucleic acid obtained, a solid phase phosphoramidite method is preferable.

A preferable embodiment of the nucleic acid synthesis reaction by a solid phase phosphoramidite method includes, for example, a method consisting of each of the following steps:

-   (a) a step of placing the support for solid phase synthesis of     nucleic acid of the present invention in a reaction column of a     nucleic acid automatic synthesizer; -   (b) a step of flowing an acid such as dichloroacetic acid solution     and the like in the reaction column to remove the     hydroxymethyl-protecting group and wash the support; -   (c) a step of sequentially performing respective steps of coupling     to bind nucleoside phosphoramidite corresponding to the 3′ terminal,     which is activated by tetrazole, to the aforementioned hydroxymethyl     group, capping of unreacted hydroxy group, and oxidation of     phosphite, and repeating the series of steps until the object     sequence is obtained; and -   (d) a step of immersing the support for solid phase synthesis of     nucleic acid to cleave the linkage moiety after completion of the     synthesis step by the apparatus, thereby affording the object     nucleic acid.

As a result of the aforementioned production method, a chemical reaction shown by the following formula occurs, and a nucleic acid having a phosphate group at the 3′ terminal is produced. To be specific, in the nucleic acid synthesis method using support (A) for solid phase synthesis of nucleic acid, which is shown in Example 1 as a preferable embodiment of the present invention, the chemical reaction shown by the following formula occurs, and a nucleic acid having a phosphate group at the 3′ terminal is produced.

In addition, in the nucleic acid synthesis method using support (B) for solid phase synthesis of nucleic acid of the present invention, which is shown in Example 4 as a preferable embodiment of the present invention, a chemical reaction shown by the following formula occurs, and a nucleic acid having a phosphate group at the 3′ terminal is produced.

Examples of the activating agent to be used in the nucleic acid production method of the present invention include, but are not limited to, 1H-tetrazole, 4,5-dicyanoimidazole, 5-ethylthio-1H-tetrazole, benzimidazolium triflate (BIT), N-phenylbenzimidazoliumtriflate, imidazoliumtriflate (IMT), N-PhIMP, 5-nitrobenzimidazoliumtriflate, triazoliumtriflate, 1-hydroxybenzotriazole (HOBT), N-(cyanomethyl)pyrrolidinium tetrafluoroborate and the like.

EXAMPLES

The present invention is explained in more detail in the following by referring to Examples, which are not to be construed as limitative.

Example 1 Production of Support (A) for Nucleic Acid Solid Phase Synthesis

2-Hydroxybenzyl alcohol (manufactured by TOKYO CHEMICAL INDSUTRY Co., Ltd.) was dissolved in pyridine, 4,4′-dimethoxytritylchloride was added, and the mixture was reacted at room temperature for 22 hr to protect the hydroxy group by a dimethoxytrityl group (DMTr). The yield of the compound wherein the hydroxymethyl group was protected with DMTr was 41%. The resultant product was dissolved in dichloromethane, succinic anhydride and triethylamine were added and the mixture was reacted at room temperature for 29 hr to give a compound wherein the linkage moiety of the succinyl group was bound. Then, a porous polystyrene solid phase support having a hydroxyl group (NittoPhase, manufactured by NITTO DENKO Co., Ltd. (registered trade mark)) was dispersed in acetonitrile, and the aforementioned compound, HBTU and N,N-diisopropylethylamine were added. The mixture was reacted at 28° C. for 23 hr to carry the aforementioned compound on the solid phase support. Sequentially, acetic anhydride, N-methylimidazole, pyridine, 4-dimethylaminopyridine and acetonitrile were added, and the mixture was reacted at 28° C. for 25 hr to cap an unreacted hydroxy group to give solid phase support (A) for nucleic acid synthesis of the present invention, which is represented by the following formula:

The binding amount of the linker of the present invention to the solid phase support obtained as mentioned above was 32 μmol/g.

Example 2 Synthesis of 5-Mer Thymidine Using Support (A) for Solid Phase Synthesis of Nucleic Acid

The solid phase support (A) for nucleic acid synthesis of the present invention (31 mg) prepared in Example 1 was packed in a reaction column, 5-mer thymidine (5′-TTTTT-3′) was synthesized by DMT-off (method of removing 5′ terminal-protecting group) (synthesis scale 1 μmol) using an automated DNA/RNA synthesizer ABI3400 (manufactured by Applied Biosystems). After the synthesis, the solid phase support bound with DNA oligonucleotide was immersed in a 30% aqueous ammonia/ethanol (2:1) mixed solution at 55° C. for 14 hr to cleave the DNA oligonucleotide from the solid phase support.

Example 3 Synthesis of 20-Mer DNA Oligonucleotide Using Support (A) for Nucleic Acid Solid Phase Synthesis

In the same manner as in Example 2, 20-mer DNA oligonucleotide (5′-ATA CCG ATT AAG CGA AGT TT-3′:SEQ ID NO: 1) was synthesized by DMT-off (synthesis scale 1 μmol) using solid phase support (A) for nucleic acid synthesis of the present invention. After synthesis, the DNA oligonucleotide was cleaved from the solid phase support.

Comparative Example 1

Synthesis of 5-Mer Thymidine Using Support for Solid Phase Synthesis Bound with DMT-dT-3′-Succinate

In the same manner as in Example 1, cleavable linker DMT-dT-3′-succinate (manufactured by Beijing OM Chemicals) was bound with a commercially available solid phase support NittoPhase (registered trade mark) (manufactured by NITTO DENKO Co., Ltd.). The binding amount of the compound to the solid phase support was 38 μmol/g. The solid phase support (27 mg) was packed in a reaction column, 5-mer DNA oligonucleotide (5′-TTTTT-3′) was synthesized by DMT-off in the same manner as in Example 1 (synthesis scale 1 μmol). After synthesis, the DNA oligonucleotide was cleaved from the solid phase support.

Comparative Example 2

Synthesis of 20-Mer DNA Oligonucleotide Using Support for Solid Phase Synthesis Bound with DMT-dT-3′-Succinate

20-mer DNA oligonucleotide (5′-ATA CCG ATT AAG CGA AGT TT-3′:SEQ ID NO: 1) was synthesized by DMT-off (synthesis scale 1 μmol) using the solid phase synthesis support bound with a cleaving linker DMT-dT-3′-succinate, which was produced in Comparative Example 1. After synthesis, the DNA oligonucleotide was cleaved from the solid phase support.

Experimental Example 1

The DNA oligonucleotide solutions obtained in Examples 2 and 3, as well as Comparative Examples 1 and 2 were measured by high performance liquid chromatography (HPLC) (measurement conditions: column; Clarity 3 μm Oligo-RP 50×4.6 mm (manufactured by Phenomenex), UV detection; 260 nm, Buffer A; 100 mM TEAA (pH 7.0), Buffer B; acetonitrile). FIG. 1A-D show each HPLC chart.

In addition, the solutions obtained in Examples 2 and 3, as well as Comparative Example 1 and 2 were subjected to high performance liquid chromatography mass spectrometry (LC-MS analysis) (measurement conditions, column; Clarity 3 μm Oligo-RP 50×4.6 mm (manufactured by Phenomenex), UV detection; 254 nm, Buffer A; 100 mM TEAA (pH 7.0), Buffer B; acetonitrile).

As a result, the main peak of the DNA oligonucleotide produced in Example 2 was confirmed to be T-5-mers having a phosphate group at the 3′ terminal (molecular weight (measured value); 1539). Moreover, the main peak of the nucleic acid produced in Example 3 was confirmed to be 20-mer oligonucleotide having a phosphate group at the 3′ terminal (molecular weight (measured value); 6219). On the other hand, the main peaks of the DNA oligonucleotides produced in Comparative Examples 1 and 2 were confirmed to be T-5-mers having 3′ terminal-OH group (molecular weight (measured value); 1459) and 20-mer DNA oligonucleotide having 3′ terminal-OH group (molecular weight (measured value); 6139), respectively.

Example 4 Production of Support (B) for Solid Phase Synthesis of Nucleic Acid

4-Hydroxybenzyl alcohol (manufactured by TOKYO CHEMICAL INDUSTRY Co., Ltd.) was dissolved in pyridine, 4,4′-dimethoxytritylchloride was added and the mixture was reacted at room temperature for 14 hr to protect the hydroxy group with the dimethoxytrityl group (DMTr). The yield of the compound wherein the hydroxymethyl group was protected with DMTr was 93%. The resultant product was dissolved in dichloromethane, succinic anhydride and triethylamine were added and the mixture was reacted at room temperature for 31 hr to give a compound wherein the linkage moiety of succinyl group is bound (yield 80%). Then, NittoPhase (registered trade mark) (manufactured by NITTO DENKO Co., Ltd.), which is a cross-linking polystyrene solid phase support having a hydroxy group was dispersed in acetonitrile, the aforementioned compound, HBTU and N,N-diisopropylethylamine were added and the mixture was reacted at 28° C. for 23 hr to carry the aforementioned compound on the solid phase support. Then, acetic anhydride, N-methylimidazole, pyridine, 4-dimethylaminopyridine and acetonitrile were added and the mixture was reacted at 28° C. for 23 hr to cap the unreacted hydroxy group, whereby solid phase support (B) for nucleic acid synthesis of the present invention, which is represented by the following formula:

was obtained.

The binding amount of the linker of the present invention to the solid phase support obtained as mentioned above was 29 μmol/g.

Example 5 Synthesis of 5-Mer Thymidine Using Support (B) for Nucleic Acid Solid Phase Synthesis

The solid phase support (B) for nucleic acid synthesis of the present invention (34 mg) prepared in Example 4 was packed in a reaction column, DNA oligonucleotide 5-mer thymidine (5′-TTTTT-3′) was synthesized by DMT-on (method of not removing 5′ terminal-protecting group) (synthesis scale 1 μmol) using an automated DNA/RNA synthesizer ABI3400 (manufactured by Applied Biosystems). After the synthesis, the solid phase support bound with DNA oligonucleotide was immersed in a 30% aqueous ammonia/ethanol (1:1) mixed solution at 55° C. for 15 hr to cleave the DNA oligonucleotide from the solid phase support.

Example 6 Synthesis of 20-Mer DNA Oligonucleotide Using Support (B) for Nucleic Acid Solid Phase Synthesis

In the same manner as in Example 5, 20-mer DNA oligonucleotide (5′-ATA CCG ATT AAG CGA AGT TT-3′:SEQ ID NO: 1) was synthesized by DMT-on (synthesis scale 1 μmol) using solid phase support (B) for nucleic acid synthesis of the present invention. After synthesis, the DNA oligonucleotide was cleaved from the solid phase support.

Comparative Example 3

Synthesis of 5-Mer Thymidine Using Support for Solid Phase Synthesis Bound with DMT-dT-3′-Succinate

A solid phase support (25 mg) bound with DMT-dT-3′-succinate produced in Comparative Example 1 was packed in a reaction column and, in the same manner as in Example 5, DNA oligonucleotide 5-mer thymidine (5′-TTTTT-3′) was synthesized by DMT-on (synthesis scale 1 μmol). After synthesis, the DNA oligonucleotide was cleaved from the solid phase support.

Comparative Example 4

Synthesis of 20-Mer DNA Oligonucleotide Using Support for Solid Phase Synthesis Bound with DMT-dT-3′-Succinate

A solid phase support (25 mg) bound with DMT-dT-3′-succinate produced in Comparative Example 1 was packed in a reaction column and, in the same manner as in Example 6, 20-mer DNA oligonucleotide (5′-ATA CCG ATT AAG CGA AGT TT-3′:SEQ ID NO: 1) was synthesized by DMT-on (synthesis scale 1 μmol). After synthesis, the DNA oligonucleotide was cleaved from the solid phase support.

Experimental Example 2

The DNA oligonucleotide solutions obtained in Examples 5 and 6, as well as Comparative Examples 3 and 4 were measured by HPLC. The measurement conditions were column; XBridge OST C18 2.5 μm 50×4.6 mm (manufactured by Waters), UV detection; 260 nm, Buffer A; 100 mM HFIP/7 mM TEA/water (pH 8.0), Buffer B; methanol. FIG. 2A-D show each HPLC chart.

In addition, the solutions obtained in Examples 5 and 6, as well as Comparative Examples 3 and 4 were subjected to LC-MS analysis (the measurement conditions were XBridge OST C18 2.5 μm 50×4.6 mm (manufactured by Waters), UV detection; 254 nm, Buffer A; 100 mM HFIP/7 mM TEA/water (pH 8.0), Buffer B; methanol).

As a result, the main peak of the nucleic acid produced in Example 5 was confirmed to be T-5-mers having a phosphate group at the 3′ terminal (molecular weight (measured value); 1840). The main peak of the DNA oligonucleotide produced in Example 6 was confirmed to be 20-mer oligonucleotide having a phosphate group at the 3′ terminal (molecular weight (measured value); 6521).

On the other hand, the main peaks of the DNA oligonucleotides produced in Comparative Examples 3 and 4 were confirmed to be T-5-mers having 3′ terminal-OH group (molecular weight (measured value); 1760) and 20-mer DNA oligonucleotide having 3′ terminal-OH group (molecular weight (measured value); 6441), respectively.

Table 1 collectively shows the data obtained in Experimental Examples 1 and 2.

TABLE 1 solid phase molecular weight support synthesis DMT (measured value) Exper. Ex. 2 (A) T-5-mers off 1539 Ex. 1 Ex. 3 (A) Mix 20-mers off 6219 Comp. DMT-dT-3′- T-5-mers off 1459 Ex. 1 succinate Comp. DMT-dT-3′- Mix 20-mers off 6139 Ex. 2 succinate Exper. Ex. 5 (B) T-5-mers on 1840 Ex. 2 Ex. 6 (B) Mix 20-mers on 6521 Comp. DMT-dT-3′- T-5-mers on 1760 Ex. 3 succinate Comp. DMT-dT-3′- Mix 20-mers on 6441 Ex. 4 succinate

INDUSTRIAL APPLICABILITY

With the development of nucleic acid pharmaceutical products in recent years, the development of a more efficient and more functional method for synthesizing nucleic acid has been desired. A nucleic acid having a phosphate group at the 3′ terminal, which is automatically synthesized using the solid phase support for nucleic acid synthesis of the present invention can be widely used for chemical linkage reaction of nucleic acid, modification of nucleic acid utilizing 3′ phosphate group, structural study of nucleic acid and the like.

This application is based on a patent application No. 2009-242445 filed in Japan (filing date: Oct. 21, 2009), the contents of which are incorporated in full herein by this reference.

SEQUENCE LISTING FREE TEXT

-   SEQ ID NO: 1 artificially synthesized oligo 20-mers 

1. A linker for solid phase synthesis of nucleic acid, comprising a compound represented by at least one of the following formulae

wherein X is a hydroxyl-protecting group dissociated by acid, L is a linkage moiety cleaved by alkali, and R¹, R², R³ and R⁴ are each independently a hydrogen atom, an alkyl group having a carbon number of 1-6, an alkoxy group having a carbon number of 1-6, an alkylamino group having a carbon number of 1-6 or a halogen atom.
 2. The linker according to claim 1, wherein L is a succinyl group.
 3. The linker according to claim 1, wherein X is a dimethoxytrityl group.
 4. A support for solid phase synthesis of nucleic acid, comprising a structure shown by at least one of the following formulae

wherein X is a hydroxyl-protecting group dissociated by acid, L is a linkage moiety cleaved by alkali, Sp is a solid phase support, and R¹, R², R³ and R⁴ are each independently a hydrogen atom, an alkyl group having a carbon number of 1-6, an alkoxy group having a carbon number of 1-6, an alkylamino group having a carbon number of 1-6 or a halogen atom.
 5. The support according to claim 4, wherein L is a succinyl group.
 6. The support according to claim 4, wherein X is a dimethoxytrityl group.
 7. The support according to claim 4, wherein Sp is a solid phase support comprised of porous synthetic polymer particles or porous glass particles.
 8. A method of producing a nucleic acid, comprising a step of performing a nucleic acid synthesis reaction on a support for solid phase synthesis of nucleic acid, the support comprising a structure shown by at least one of the following formulae

wherein X is a hydroxyl-protecting group dissociated by acid, L is a linkage moiety cleaved by alkali, Sp is a solid phase support, and R¹, R², R³ and R⁴ are each independently a hydrogen atom, an alkyl group having a carbon number of 1-6, an alkoxy group having a carbon number of 1-6, an alkylamino group having a carbon number of 1-6 or a halogen atom.
 9. The production method of claim 8, wherein L is a succinyl group.
 10. The production method of claim 8, wherein X is a dimethoxytrityl group.
 11. The production method of claim 8, wherein the nucleic acid synthesis reaction is performed by a solid phase phosphoramidite method. 